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3D BIOPRINTING FOR VASCULAR STRUCTURES

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INTRODUCTION

The WHO defines health as “A state of complete physical, mental and social wellbeing, and not merely the absence
of any disease or infirmity.” The failure of physical health is, at most times, one of the major causes of death or
debilitation. The idea of replaceable, artificially engineered, biocompatible body parts and organs, which might have been a pipe dream for every science fiction lover, has gradually emerged as one of the front-runners in research over the last half a decade. In a world where there is a gross variation in the kind of lifestyle from person
to person, there is a need for customizable parts that are both compatible and also suit the specific requirements
of the individual. Three-dimensional printing provides precise control, and can be used with a large number of
biological structures. Various organs can be printed and sustained in a lab grown environment, of which the tissue
level in two dimensions is the easiest to achieve and can be used in drug interaction testing. However, the end
game in the field is the successful bioprinting of three-dimensional organ level structures with adequate
vasculature, as cells that are more than around 200 micrometers — a couple of human-hair widths — away from a
source of nutrients quickly die. Therefore, creating a realistic vascular system will be necessary if researchers are to
successfully build organs. Such a system can sustain the organs independent of a lab, and can be used for organ transplants; which, as futuristic as it sounds, could one day result in quasi-immortality; as all failing organs could be replaced as and when required to further prolong one’s life as long as one wishes to.

OBJECTIVES

To outline the progression in the field of vascular bioprinting

To identify the revolutionary researchers in the field

To establish the potential uses of current trends

To identify the complications with the current technologies

To recognize the future needs and potential of the technology

PROGRESSION OF THE TECHNOLOGY

Considering a timeline extending from 2012 up until the time of publishing, one of the earliest attempts at creating a branched, bio-printed vascular system was made by researchers at the University of Penn in 2012, by using a
rapid casting method to create 3D-printed templates of filament networks that used a combination of sucrose and
glucose along with dextran for structural reinforcement and printed with RepRap, an open-source 3D printer with
a custom-designed extruder and controlling software.[1]

2015 was a watershed year in the current field- various newer technologies, methods and bio-inks were created and, for the first time, a 3D printed vascular structure was successfully implanted in rat aortae, in a collaboration
between Saga University and Cyfuse biomedical, Japan, using their patented 3D bioprinter known as Regenova.
They used a method where Human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells
(HASMC) and human normal dermal fibroblasts (HNDFB) were cultured in an appropriate medium. The resultant
multicellular spheroids (MCSs) were used to assemble MCSs for constructing scaffold-free tubular tissue. Upon
perfusion with a bioreactor, a tubular tissue of 1.5 mm in diameter and 7 mm in length was obtained, which was
then implanted into the abdominal aorta of nude rats. The flow rate was monitored and found adequate, and
furthermore, the graft compliance was noted and the rats were euthanized.
[2]

Using an inkjet 3D printer with the droplet dispersion method, researchers from the Rensselaer Polytechnic Institute, New York and Harvard Medical school, Boston, cultured Human umbilical vein endothelial cells (HUVEC’s); and collagen hydrogel precursor (Rat tail, type I) was used as a scaffold material. The fabricated vasculature has a
tight, confluent endothelium lining, presenting barrier function for both plasma proteins and high-molecular
weight dextran molecules, but could only sustain tissue viability for a distance of up to 5mm under simulated
human body conditions. [3]

EU research project ArtiVasc 3D, which comprised an interdisciplinary team of more than 20 partners and
researchers led by the Fraunhofer Institute for Laser Technology, combined the freeform methods of inkjet printing
and stereo lithography. With these combined processes, the researchers were able to achieve a very fine
resolution for the construction of branched, porous blood vessels with layer thicknesses of about 20 microns. The
researchers used mathematical simulations to develop data for the construction of branched structures, to allow
for uniform blood flow. [4]

Using a process called Electrospinning, Techshot Inc., in collaboration with the Pentagon’s defense health program,
have created implantable vascular grafts with stable vascular features. Stem cells collected from the patient’s own
fat are printed onto a tubular scaffold. Upon complete healing, within six to nine months, the polymer tube is
absorbed by the body and a natural blood vessel is left behind. This research was carried out mainly to treat war
wounds of soldiers with severe injuries to their extremities. Pre-clinical trials have been completed and human
clinical trials for this invention are expected to begin in the last quarter of 2017. [5]

Researchers at the Lawrence Livermore National Laboratory used a combination of 3D printing and biologically
compatible material, and the environment is engineered to enable human capillaries to develop on their own. This
process takes a while, so initially, tubes are printed out of cells and other biomaterials to deliver essential nutrients
to the surrounding printed environment. Eventually, the self-assembled capillaries are able to connect with the
bio-printed tubes and deliver nutrients to the cells on their own, enabling these structures to function like they do
in the body, resembling a “spaghetti bowl.” This research leverages the body's ability for self-directed growth, and
the result is something rather akin to human physiology. [6]

A team at Harvard University in Cambridge, Massachusetts, printed thick tissue with a rudimentary vascular system
and have managed to keep it alive for weeks. The team used three different inks: silicone to give a basic shape; a
bio ink infused with pluripotent stem cells that would turn into the tissue; and Pluronic, which is a gel at room
temperature, but a liquid when cooled. They successfully printed tissue which was 1 centimeter thick and kept it
alive for a period of over 6 weeks. While the vasculature for this was rudimentary, the same principle can be
applied to create more complex vascular networks. [7]

In 2016, Cyfuse Medical introduced a newer 3D bio printing method called the Kenzan Method, a unique form of bio printing that uses tiny vertical spikes on which cell clusters (spheroids) are skewered and kept in position, rather than use a bio ink. The Saga University scientists believe that their 3D bio printing method could help treat patients recovering from myocardial infarctions. [8]

For the first time, at the end of 2016, cardiac and vascular structures were 3D-printed in a zero gravity environment, using adult human stem cells; by a team of scientists working for the NASA, and as a collaboration between SpaceX contractor Techshot, bio printing experts nScrypt and bio-ink innovators Bioficial Organs. The
project saw researchers take to the skies in a Zero Gravity Corporation parabolic flight, to test out the printer
prototype. The combined expertise resulted in a bioprinter capable of printing extremely fine lines in space. The
major advantages to this method are: firstly, the ability to use finer print tips and lower viscosity bio-inks (which
enable faster printing) that contain only the biological materials needed to create a healthy organ; and secondly,
the absence of gravity enables easier printing of hollow structures, which do not collapse upon themselves. 3D
printing in space has the potential to be the biggest game changer in this field, as it could be the answer to both
the organ shortage and creating vasculature for printed organs. The technology can also be used to enable people
to stay longer in space. [9][10]

Researchers at the University of California, San Diego in the Nano biomaterials, Bio printing, and Tissue Engineering
Laboratory directed by Prof Shaochen Chen, have successfully manufactured branched vasculature, and the
technology used has resulted in a network featuring branching vessels that become successively smaller, similar to
those found in living tissue. The method involves printing tiny (4 millimeters × 5 millimeters, 600 micrometers
thick), pre-designed vascular networks that turn into vessel-like structures made of the same cells as natural blood
vessels. The new method, called microscale continuous optical bio printing (μCOB), involves embedding a mix of
different kinds of live cells into a hydrogel and then using ultraviolet light and mirrors to heat up and solidify 3D
patterns within the hydrogel in vivo. While they are able to transmit blood, they are currently still unable to
transport waste and other nutrients.[11][12]

Researchers in China have successfully transplanted blood vessels into Rhesus monkeys after bio printing them. A
special kind of bio-ink, being marketed as “Biosynsphere,” which is derived from stem cells acquired from fat tissue
of the monkeys was used; this is safer and has low risk of rejection. A team led by Dr. Kang Yujian, replaced a 2
centimeter part of the abdominal artery in 30 Rhesus monkeys. The stem cells were able to grow all the required
cells for a blood vessel. Post-surgical observation after a few months showed complete integration with preexisting vasculature. The ability of Biosynsphere to develop collagen is a major breakthrough in the field of printing
vascular structures. Sichuan Revotek manufactured the ink to be compatible with their blood vessel-printing 3D
bioprinter that uses cloud computing; and together, they have the ability to completely overhaul the vascular 3D
printing industry. [13][14]

COMPANIES AND THEIR FUNDING

Unsurprisingly, China and Japan lead the charts in terms of raising funding, increased research as well as resultant
patent filings. One of the major players is Cyfuse, erstwhile known as CyfuseBio, which has collaborated with Saga
University, based out of Japan, in developing a 3D printer known as Regenova, which uses the Kenzan method. The
Kenzan method was invented by prof. Koich Nakayama (Saga University) and is a globally patented intellectual
property. The projects are funded by a mixture of private and public funding; up to 2 billion Yen was raised by 12
investors, including venture capital funds and corporate investors. [15] An undisclosed sum is also sponsored by the
Japan Agency for Medical Research and Development (AMED).

Sichuan Revotek, a Chinese company, is the only company to have developed a 3D printer made especially
customizable to the printing of blood vessels, using a specifically made bio-ink. Based out of Chengdu in China, the
health care startup has raised 33 million dollars in funding from a private real estate firm that wished to branch
out. [14]

In general, Chinese provinces have created unique zones where new business ventures, especially technology
startups and medical research firms, can seek funding opportunities due to a country-wide campaign to encourage
entrepreneurship. Extensive funding opportunities and an abundance of highly qualified manpower explain their
dominance in the field.

Academically, the University of California, San Diego campus hass been a spearhead in the field. They were granted
a 1.5 million dollar grant in 2015 from the NIH, specifically for the purpose of recreating vascularized tissue. [16]
This grant, along with other private investments, has helped UC along in the goal.

As recently as April 2017, Dr. Yi Hong, engineer and assistant professor in the University Of Texas at Arlington, from the Department of Bioengineering, has been awarded a grant. Dr. Hong received an R21 grant from the National
Institute of Health (NIH), for $211,000, to develop materials to make 3D-printed blood vessels for children suffering from vascular defects. Dr. Hong, who has been on research grants totaling over $850,000 in his career, will develop
materials that are elastic and can be formed into patient-specific, feasible blood vessels that can also be used in a
3D printer. Dr. Guohao Dai, who is his partner at Northeastern University, will 3D-print the blood vessels using Dr.
Hong’s materials. [17]

Bio 3D-printing of vasculature in space is a collaboration between NASA, SpaceX contractor Techshot (which
provides the vehicle or craft necessary for the parabolic flight), bioprinting experts nScrypt (who manufactured the
3D printer to be specifically compatible for space) and bio-ink innovators Bioficial Organs. The details of the funds
involved are a closely guarded secret, due to the project’s association with the government.

Other companies and universities working in this area are:

ArtiVasc 3D, by the Fraunhofer Institute for Laser Technology ILT, sponsored by over 20 companies and
subsidized under adequate government grants.

Cellink, a Swedish company that manufactures bio printers and bioinks. Completely customizable printers can
be assembled by them. The company is both privately owned and funded, with the major share being shared
between Erik Gatenholm (40.4%) and Hector Daniel Avila Martinez (26.8%).

Rokit, a South Korean company, was recently given a $3 million government grant. The company collaborating
with the Korean Institute of Science and Technology (KIST), Seoul National University, Bundang Hospital,
Hanyang University, and Korea Institute of Machinery and Materials (KIMM).

BioBots, a startup, which recently raised over $300,000 on equity crowdfunding platform FundersClub, is
concentrating mainly on manufacturing affordable desktop 3D bioprinters. However, the company is well
renowned for its bioinks, priced at a 1000$, which can be used with other printers as well.

OBSTACLES

1. At a vascular level:

The major obstacle to bioprinting vasculature is recreating the micro vessels, especially at the capillary
level, including the extensive branching that is naturally seen.

To exactly replicate the strength, ability to maintain lumen patency, viability and elasticity of natural
vessels, even when empty.

To recreate vasculature that can be repaired if required, and their ease of attachment into the existing
system in order to maintain continuity.

2. At the organ level:

There are multiple contrasting theories for printing vasculature. While some debate that it is easier to
print a larger structure to serve as an organ leaving hollows for the vessels, the converse has also been
argued. Both methods have attained a modicum of success so far, but there remains to be seen the
development of a method that can manage to print organs with vessels, successfully and viably.

The life of 3D-printed vessels has so far only been managed up to 6 weeks. However, for the purpose to
be fruitful, the vessels should have the ability to survive and regenerate as the surrounding vessels do.
This is especially essential if the technology has to be used to vascularize entire organ systems.

Tissue death occurs at a distance of 200 micrometers from the nearest source of blood supply. The lack of
technology in creating that level of microcirculation is the biggest hindrance to the progression of de novo
organ printing. An organ cannot survive without adequate vascularization, hence there is a great need for
technology that enables the printing of patent, viable capillaries vasculature, including capillaries.

To attain organ level vascularization, the circulation needs to be at a three-dimensional level. So far, the
technology has only been able to achieve vascularization at a two-dimensional level.

THE FUTURE SCENARIO

3D-printing of the entire vasculature of a patient pre-operatively, so as to be able to pre-emptively prevent
any surgical complications is a current application. Using CT and MRI-imaging scans to recreate three
dimensional images and printing them out to accurately recreate the vascular structures, thus enabling
surgeons to precisely treat aneurysms and the like.

Bioprinted vessels can be made, in a way as to avoid blockage completely, with the addition of antithrombolytic structures or materials. Another method being tested is the usage of acellular materials and
natural polymer composition, which through multiple modes can be used to form modified blood vessels.
Currently, the adaptability of materials like collagen, silk fibroin or polylactic acid is being tested. [18] Such
vessels may very well be lifesaving in the case of patients with hypertension and those suffering from cardiac
conditions.

Organ system integration needs to be achieved. It does not suffice to merely have a vascular system for an
organ; the real test lies in the ability of bio printed vessels to integrate and bond with the preexisting
vasculature.

Fully functional, viable vasculature, as close to nature as possible, which are also affordable as a technology;
and those that do not cause any adverse reactions, fatal or otherwise, is something that needs to be worked
on with urgency.

Customizable vascular structures, which can be made according to patients’ requirements, printed using cells
from the patient, are operable, can be replaced or repaired with ease in the future, and do not require an
extremely long time to be created, or have long waiting lists for the procedure, will essentially bring about a
lot of changes in the health care industry; especially in battle scenarios and road traumas; where time is of the
essence.

The most anticipated development from said technology is integrating a fully vascularized, viable, printed
organ system into an animate body and having it carry on functioning as any other biological tissue. This
development will eliminate the need for transplant lists, waiting for organ donors, deaths caused due to lack
of organs donated; and may also completely wipe out the thriving illegal organ-trading industry.

CONCLUSION

All things considered, this field of study is only growing exponentially. Newer advancements in 3D printers, bioinks, adjuvant materials, etc. are coming in hard and fast. While alternative methods; such as the recent research
by the Worcester Polytechnic Institute (WPI), which involved decellularising a spinach leaf and perfusing it with
blood using the plant’s own vascular system, resulting, over a course of 21 days, in functional contractile heart
tissue;[19] have their merits, the customization that can be achieved with 3D-printing is unparalleled.

A future in which everyone can live a life of longevity, renewability and sustained good health, is not far-off. While everlasting life may not be everyone’s cup of tea, the ability to live a healthy life, for as long as one is alive, is definitely something to ponder. This is not to say that athanasia can be achieved, or that it is even a morally acceptable scenario; however, a world of mostly able-bodied individuals, as opposed to people being indisposed, is not something to scoff at. A world may soon be changed, as science and technology grows in leaps and bounds; all that now remains to be seen is whether the monkeys and the rats win the race.

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